Project Summary/Abstract ? Project 1 The long-term goals of the research of this project are 1) to define the mechanisms by which herpes simplex virus 1 (HSV-1) gene products promote euchromatin on viral lytic genes during lytic infection and heterochromatin on these genes during latent infection of sensory neurons to determine the switch between lytic and latent infection, and 2) to apply this knowledge to develop therapeutics for HSV latent infection. Antiviral drugs that inhibit HSV lytic infection have been developed, but there is no antiviral therapeutic for latent infection. Previously, we have defined many aspects of HSV latent infection of sensory neurons in murine trigeminal ganglia, in particular the structure of latent HSV-1 chromatin and the viral gene products that regulate viral chromatin structure. In the proposed research, we will test mechanisms of latent infection in the murine trigeminal ganglion model, in cultured mouse neurons and, for greater medical relevance, in human inducible pluripotent stem cell-derived neurons. We will focus on functions of viral gene products that promote a stable latent infection that is poised for reactivation. All aims are integrated with Projects 2 and 3 and Cores A and B. Specific Aim 1 will test various hypotheses about how the latency-associated transcript (LAT) promotes total histone and facultative heterochromatin loading on latent HSV-1 DNA. We will test whether LAT promotes epigenetic silencing by antisense transcription by inserting transcriptional terminators in the LAT transcriptional unit in the HSV-1 genome, and testing the effects on chromatin, transcripts antisense to ICP4, and expression of downstream antisense genes. We will test if LAT recruits histone loaders and epigenetic factors to the HSV-1 genome to promote loading of heterochromatin to the viral genome and stable maintenance of poised latency. Specific Aim 2 will explore mechanisms by which ICP0 promotes stable maintenance of the latent HSV-1 genome. We will determine the role of ICP0 in maintaining the viral genome, heterochromatin, and LAT expression in human neurons, murine neurons, and murine in vivo systems. The effects of a novel, exciting ICP0-specific inhibitory small molecule will be defined in murine and human neuronal latency systems. Specific Aim 3 will study the mechanism of action of the CCCTC-binding factor (CTCF) binding site (CTRL2) between the LAT and ICP0 gene promoters, which serves as a chromatin insulator and promotes reactivation. We will define the role of CTCF binding at the at the CTRL2 site on establishment and maintenance of latent infection. We will define the role of CTRL2 in the 3-dimensional structure of viral chromatin in human and murine neurons with Project 3. These studies will provide important new basic knowledge of the mechanisms of HSV latent infection and may enable new therapeutics for the HSV latent infection.